EP0785950B1 - Analogen des keratinozytenwachstumfaktors - Google Patents
Analogen des keratinozytenwachstumfaktors Download PDFInfo
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- EP0785950B1 EP0785950B1 EP95938216A EP95938216A EP0785950B1 EP 0785950 B1 EP0785950 B1 EP 0785950B1 EP 95938216 A EP95938216 A EP 95938216A EP 95938216 A EP95938216 A EP 95938216A EP 0785950 B1 EP0785950 B1 EP 0785950B1
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- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/475—Growth factors; Growth regulators
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Definitions
- the present invention relates to recombinant DNA technology and protein engineering. Specifically, recombinant DNA methodologies have been applied to generate polypeptide analogs of keratinocyte growth factor (KGF), a potent mitogen of non-fibroblast epithelial cell growth, wherein the analogs have improved stability as compared to that of the parent KGF.
- KGF keratinocyte growth factor
- soft tissue growth factors protein factors sometimes referred to as soft tissue growth factors. These molecules are generally released by one cell type and act to influence proliferation of other cell types. (Rubin et al. (1989), Proc. Nat'l. Acad. Sci. USA, 86 :802-806). Some soft tissue growth factors are secreted by particular cell types and influence the proliferation, differentiation and/or maturation of responsive cells in the development of multicellular organisms (Finch et al. (1989), Science, 245 :752-755). In addition to their roles in developing organisms, some are significant in the continued health and maintenance of more mature systems. For instance, in mammals there are many systems where rapid cell turnover occurs. Such systems include the skin and the gastrointestinal tract, both of which are comprised of epithelial cells. Included within this group of soft tissue growth factors is a protein family of fibroblast growth factors (FGFs).
- FGFs fibroblast growth factors
- FGF family members which share a relatedness among primary structures: basic fibroblast growth factor, bFGF (Abraham et al . (1986), EMBO J., 5 :2523-2528); acidic fibroblast growth factor, aFGF (Jaye et al . (1986), Science, 233 :541-545); int-2 gene product, int-2 (Dickson & Peters (1987), Nature , 326 :833); hst/kFGF (Delli-Bovi et al. (1987), Cell, 50 :729-737 and Yoshida et al. (1987), Proc. Natl. Acad. Sci.
- FGF-5 Zhan et al. (1988), Mol. Cell. Biol., 8 :3487-3495
- FGF-6 Marics et al . (1989), Oncogene, 4 :335-340
- keratinocyte growth factor Finch et al . (1989), Science, 24 :752-755
- hisactophilin Habazzettl et al . (1992), Nature, 359 :855-858.
- KGF keratinocyte growth factor
- hKGF human
- rKGF recombinant polypeptide
- amino acid numbering for molecules described herein shall correspond to that presented for the mature form of the native molecule (i.e., minus the signal sequence), as depicted by amino acids 32 to 194 of SEQ ID NO:2.
- Native KGF may be isolated from natural human sources (hKGF) or produced by recombinant DNA techniques (rKGF) (Finch et al . (1989), supra; Rubin et al . (1989), supra; Ron et al . (1993), The Journal of Biological Chemistry, 268(4) :2984-2988; and Yan et al . (1991), In Vitro Cell. Dev. Biol., 27A :437-438).
- hKGF natural human sources
- rKGF recombinant DNA techniques
- native KGF is relatively unstable in the aqueous state and that it undergoes chemical and physical degradation resulting in a loss of biological activity during processing and storage (Chen et al . (1994), Pharmaceutical Research, 11 :1582-1589).
- Native KGF is prone also to aggregation at elevated temperatures and it becomes inactivated under acidic conditions (Rubin et al. (1989), Proc. Natl. Acad. Sci. USA, 86 :802-806). Aggregation of native KGF in aqueous solution also results in inactivated protein. This is disadvantageous because such loss of activity makes it impractical to store aqueous formulations of native KGF proteins for extended periods of time or to administer the protein over extended periods.
- bFGF and aFGF have been modified by deleting or substituting positively-charged residues, which are important for heparin binding with neutral or negatively-charged amino acids. It was reported that the modified molecules resulted in reduced heparin binding activity. Accordingly, it was taught that the amount of modified molecule sequestered by heparin and/or heparin-like molecules in a patient would be reduced, thereby increasing potency as more of the FGF will reach its targeted receptor (EP 0 298 723).
- the literature has not reported a modified KGF molecule having significantly improved stability relative to native KGF. Moreover, the literature has not reported sufficient teachings or evidence to provide a reasonable expectation of successfully generating KGF molecules with such desirable characteristics.
- the effects upon biological activity of any amino acid change upon the protein will vary depending upon a number of factors, including the three-dimensional structure of the protein and whether or not the modification is to either the heparin binding region or the receptor binding region on the primary sequence of the protein.
- the knowledge within the art does not permit generalization about the effects of amino acid modifications to native KGF based upon the effects of amino acid modifications on even commonly categorized proteins.
- the present invention provides novel, biologically active polypeptide analogs of native keratinocyte growth factor termed "KGF," wherein native KGF corresponds to the following sequence said analogs having an improved stability wherein the analogs comprise the amino acid sequence of KGF having a charge-change by the deletion and/or substitution of one or more of amino acid residues 41-154 of the above sequence to effect a reduced positive charge as compared with native KGF, optionally further having a deletion of the first 15 to 24 amino acids of the N-terminal native KGF, having residues corresponding to Cys (1) and Cys (15) replaced or deleted, having an N-terminal methionine or having a signal sequence, with the proviso that the amino acid sequence 123-131 of the above sequence is not substituted by any one of the amino acid sequences selected from the group of DLYQG and AKYEG.
- KGF native keratinocyte growth factor
- the resultant KGF analog has improved stability as compared to the parent molecule.
- the invention is directed to those analogs which also exhibit full biological activity (i.e., at least substantially similar receptor binding or affinity) as compared to native KGF.
- nucleic acid molecules encoding the various biologically active polypeptide analogs of KGF are described.
- nucleic acids comprise DNA molecules cloned into biologically functional plasmid or viral vectors.
- nucleic acid constructs may then be utilized to stably transform a procaryotic or eucaryotic host cell.
- the invention involves a process wherein either a procaryotic (preferably E. coli) or eucaryotic host cell stably transformed with a nucleic acid molecule is grown under suitable nutrient conditions in a manner allowing the expression of the KGF analog. Following expression, the resultant recombinant polypeptide can be isolated and purified.
- a further aspect of the invention concerns pharmaceutical formulations comprising a therapeutically effective amount of a KGF analog and an acceptable pharmaceutical carrier. Such formulations will be useful in treating patients afflicted with epithelial diseases and injuries.
- another aspect relates to the use of a KGF analog for preparing a medicament for the treatment of disease requiring the stimulation of the production of non-fibroblast epithelial cells.
- epithelial cells include various adnexal cells, pancreatic cells, liver cells, and mucosal epithelium in the respiratory and gastrointestinal tracts.
- novel analogs of KGF are provided.
- the KGF analogs are preferably produced by deleting or substituting one or more specific, positively-charged residues in KGF.
- the KGF analogs have, among other properties, an improved stability under at least one of a variety of purification and/or storage conditions.
- the KGF analogs will generally be purified in a greater yield of soluble, correctly folded protein.
- the material once the material is purified, it will be more stable to pH, temperature, etc. as compared to the stability of the parent molecule.
- arginine residue at position 144 may correspond to a residue in bFGF, as determined by X-ray crystallography, which is reported to be within or near a cluster of basic residues that mediate heparin binding (Ago, et al. (1991), J. Biochem., 110 :360-363; and Eriksson et al. (1993), Protein Science, 2 :1274-1284).
- Native KGF contains 46 charged residues, 27 of which carry a positive charge.
- a comparison of the native KGF primary sequence with the primary sequence of bFGF suggests that some of the 27 positively charged residues form a cluster similar to a cluster found in the tertiary structure of bFGF.
- substitution of one or more of these clustered residues with amino acids carrying a negative or neutral charge may alter the electrostatic interactions of adjacent residues and may be useful to achieve increased stability.
- a "KGF analog” or a "polypeptide analog of KGF” shall mean charge-change polypeptides wherein one or more of amino acid residues 41-154 (amino acids 72-185 of SEQ ID NO:2), specifically including amino acid residues 123-133 (amino acids 154-164 of SEQ ID NO:2), are deleted or substituted with a neutral residue or negatively charged residue selected to effect a protein with a reduced positive charge.
- Preferred residues for modification are Arg 41 , Gln 43 , Lys 55 , Lys 95 , Lys 128 , Asn 137 , Gln 138 , Lys 139 , Arg 144 , Lys 147 , Gln 152 , Lys 153 or Thr 154 , with Gln 138 , Lys 139 , Arg 144 , Lys 147 , Gln 152 or Lys 153 being more preferred and Arg 144 being most preferred.
- Preferred amino acids for substitution include glutamic acid, aspartic acid, glutamine, asparagine, glycine, alanine, valine, leucine, isoleucine, serine and threonine, with glutamic acid, glutamine, aspartic acid, asparagine and with alanine being particularly preferred.
- Preferred polypeptide analogs according to the present invention are those wherein the deleted or substituted amino acid residues are selected from the arginine residue at amino acid position 41, the glutamine residue at amino acid position 43, the lysine residue at amino acid position 55, the lysine residue at amino acid position 95, the asparagine residue at amino acid position 137, the glutamine residue at amino acid position 138, the lysine residue at amino acid position 139, the arginine residue at amino acid position 144, the lysine residue at amino acid position 147, the glutamine residue at amino acid position 152, the lysine residue at amino acid position 153, or the threonine residue at amino acid position 154 of the sequence depicted in claim 1.
- polypeptide analogs of the present invention are those selected from R(144)Q, C(1,15)S/R(144)E, ⁇ N23/R(144)Q, ⁇ N23/N(137)E, ⁇ N23/K(139)E, ⁇ N23/K(139)Q, ⁇ N23/R(144)A, ⁇ N23/R(144)E (a KGF analog having a deletion of the first 23 amino acids of the N-terminus and a substitution of glutamic acid for arginine at amino acid position 144 of native KGF), ⁇ N23/R(144)L, ⁇ N23/K(127)E, ⁇ N23/K(137)Q, ⁇ N23/K(153)E, ⁇ N23/K(153)Q, or ⁇ N23/Q(152)E/K(153)E.
- any modification should give consideration to minimizing charge repulsion in the tertiary structure of the molecule; most preferably the analog will have increased stability compared with the parent molecule. Obviously, the deletions or substitutions should not be so numerous nor be made to residues of such close proximity so as to set up charge repulsion between two negatively-charged residues.
- the nucleic acids encoding such polypeptides will differ in one or more nucleotides as compared to the native KGF nucleotide sequence.
- Such polynucleotides may be expressed and the resultant polypeptide purified by any one of a number of recombinant technology methods known to those skilled in the art.
- DNA sequences coding for all or part of the KGF analogs may include, among other things, the incorporation of codons "preferred" for expression in selected host cells (e.g., "E. coli expression codons”); the provision of sites for cleavage by restriction enzymes; and the provision of additional initial, terminal, or intermediate nucleotide sequences (e.g., as an initial methionine amino acid residue for expression in E. coli cells), to facilitate construction of readily expressed vectors.
- the present invention also provides for recombinant molecules or vectors for use in the method of expression of the polypeptides.
- Such vectors may be comprised of DNA or RNA and can be circular, linear, single-stranded or double-stranded in nature and can be naturally-occurring or assemblages of a variety of components, be they naturally-occurring or synthetic.
- expression vectors useful in this invention will contain at least one expression control element functionally associated with the inserted nucleic acid molecule encoding the KGF polypeptide analog. This control element is responsible for regulating polypeptide expression from the nucleic acid molecules of the invention.
- Useful control elements include, for example, the lac system, the trp system, the operators and promoters from phage ⁇ , a glycolytic yeast promoter, a promoter from the yeast acid phosphatase gene, a yeast alpha-mating factor, and promoters derived from adenovirus, Epstein-Barr virus, polyoma, and simian virus, as well as those from various retroviruses.
- a glycolytic yeast promoter a promoter from the yeast acid phosphatase gene
- yeast alpha-mating factor promoters derived from adenovirus, Epstein-Barr virus, polyoma, and simian virus, as well as those from various retroviruses.
- numerous other vectors and control elements suitable for procaryotic or eucaryotic expression are known in the art and may be employed in the practice of this invention.
- Suitable procaryotic cloning vectors may include plasmids from E. coli (e.g. pBR322, col E1, pUC, and the F-factor), with preferred plasmids being pCFM1156 (ATCC 69702), pCFM1656 (ATCC 69576) and pCFM3102 (described in the Examples section, below).
- E. coli e.g. pBR322, col E1, pUC, and the F-factor
- preferred plasmids being pCFM1156 (ATCC 69702), pCFM1656 (ATCC 69576) and pCFM3102 (described in the Examples section, below).
- Other appropriate expression vectors of which numerous types are known in the art for mammalian, insect, yeast, fungal and bacterial expression can also be used for this purpose. The transfection of these vectors into appropriate host cells can result in expression of the KGF analog polypeptides.
- Host microorganisms useful in this invention may be either procaryotic or eucaryotic.
- Suitable procaryotic hosts include various E. coli (e.g., FM5, HB101, DH5 ⁇ ,DH10, and MC1061), Pseudomonas, Bacillus, and Streptomyces strains, with E. coli being preferred.
- Suitable eucaryotic host cells include yeast and other fungi, insect cells, plant cells, and animal cells, such as COS (e.g., COS-1 and COS-7) and CV-1 monkey cell lines, 3T3 lines derived from Swiss, Balb-c or NIH cells, HeLa and L-929 mouse cells, and CHO, BHK or HaK hamster cells.
- COS e.g., COS-1 and COS-7
- CV-1 monkey cell lines 3T3 lines derived from Swiss, Balb-c or NIH cells, HeLa and L-929 mouse cells, and CHO, BHK or HaK
- the preferred production method will vary depending upon many factors and considerations; the optimum production procedure for a given situation will be apparent to those skilled in the art through minimal experimentation.
- the resulting expression product may then be purified to near homogeneity using procedures known in the art.
- a typical purification procedure for procaryotic cell production involves rupturing the cell walls by high pressure or other means, centrifugation or filtration to remove cellular debris, followed by ion exchange chromatography of supernatant or filtrate and, finally, hydrophobic interaction chromatography.
- another purification technique involves first solublizing the inclusion bodies containing the analogs followed by ion exchange chromatography, then refolding of the protein, and, finally, hydrophobic interaction chromatography.
- U.S.S.N. 08/323,339 teaches a method for purifying a keratinocyte growth factor comprising: (a) obtaining a solution comprising the KGF; (b) binding the KGF from the solution of part (a) to a cation exchange resin; (c) eluting the KGF in an eluate solution from the cation exchange resin; (d) either passing the eluate solution from part (c) through an appropriate molecular weight exclusion matrix or performing hydrophobic interaction chromatography on the eluate solution of part (c); and (e) recovering the KGF from the molecular weight exclusion matrix or hydrophobic interaction chromatography.
- the analogs may be rapidly screened to assess their physical properties.
- the Examples sets forth various well-known stability assays, although the specific assay used to test the analog is not critical.
- the level of biological activity e.g., receptor binding and/or affinity, mitogenic, cell proliferative and/or in vivo activity
- Numerous assays are well-known and can be used to quickly screen the KGF analogs to determine whether or not they possess acceptable biological activity.
- One such assay specifically tests the KGF analogs for the ability to bind to the KGF receptor (KGFR) by competing with 125 I-KGF binding (Bottaro et al. (1990), J. Biol.
- KGF analogs can be rapidly screened for their biological activity.
- the KGF analogs may be further modified to contain additional chemical moieties not normally a part of the peptide.
- Such derivatized moieties may improve the solubility, absorption, biological half life, and the like of the KGF analog.
- the moieties may alternatively eliminate or attenuate any undesirable side effects of the protein and the like. Moieties capable of mediating such effects are disclosed, for example, in REMINGTON'S PHARMACEUTICAL SCIENCES, 18th ed., Mack Publishing Co., Easton, PA (1990).
- Covalent modifications may be introduced into the molecule by reacting targeted amino acid residues of the peptide with an organic derivatizing agent that is capable of reacting with selected side chains or terminal residues (T.E.
- Polyethylene glycol is one such chemical moiety which has been used in the preparation of therapeutic protein products.
- PEG Polyethylene glycol
- the attachment of polyethylene glycol has been shown to protect against proteolysis, Sada, et al. (1991), J. Fermentation Bioengineering, 71 :137-139 , and methods for attachment of certain polyethylene glycol moieties are available. See U.S. Patent No. 4,179,337, Davis et al ., "Non-Immunogenic Polypeptides," issued December 18, 1979; and U.S. Patent No.
- the present invention is directed to a single-dose administration unit of a medicinal formulation which can be safely administered parenterally or orally to treat a disease in a warm-blooded animal (such as a human).
- a medicinal formulation may be in the form of a lyophilized or otherwise dehydrated therapeutic or diagnostic which can be reconstituted by the addition of a physiologically acceptable solvent.
- the solvent may be any media such as sterile water, physiological saline solution, glucose solution or other aqueous carbohydrates (e.g., polyols such as mannitol, xylitol, glycerol) which is capable of dissolving the dried composition, is compatible with the selected administration route and which does not negatively interfere with the active principle and the reconstitution stabilizers employed.
- the present invention is directed to a kit for producing the single-dose administration unit.
- the kit contains both a first container having a dried protein and a second container having an aqueous formulation comprising a reconstitution stabilizer.
- concentration of the protein in the solution the solution volume which is charged into each container, and the capacity of the containers (interrelated parameters which can be suitably modified, depending upon the desired concentration of active principle in the end-dosage unit), these may vary within wide ranges well-known to skilled artisans.
- KGF analogs according to the invention may be useful as therapeutic and diagnostic agents and as research reagents.
- the KGF analogs may be used in in vitro and/or in vivo diagnostic assays to quantify the amount of KGF in a tissue or organ sample or to determine and/or isolate cells which express KGFR (Bottaro et al. (1990), J. Biol. Chem., 265 :12767-12770; Ron et al. (1993), J. Biol. Chem., 268 :2984-2988).
- 125 I-KGF analog binding to KGFR there will be less radioactivity from 125 I-KGF analog binding to KGFR, as compared to a standardized binding curve of 125 I-KGF analog, due to unlabeled native KGF binding to KGFR. Similary, the use of 125 I-KGF analog may be used to detect the presence of KGFR in various cell types.
- This invention also contemplates the use of a KGF analog in the generation of antibodies made against the peptide, which antibodies also bind to native KGF.
- the antibodies are monoclonal or polyclonal in origin and are generated using a KGF analog.
- the resulting antibodies bind preferentially to native KGF, preferably when that protein is in its native (biologically active) conformation. These antibodies can be used for detection or purification of the KGF.
- the invention contemplates the use of KGF analogs in the discovery of high affinity or low affinity KGF binding molecules having therapeutical applications, for example, as a way for efficient KGF delivery or as an inhibitor for KGF activity.
- the thermal stability of the KGF analogs is important to identify such binding molecules in physiological conditions (i.e., at 37°C) since their affinity for KGF could be strongly temperature-dependent and may be unpredictable from the affinity observed at 4°C.
- the KGF analogs may be formulated with additives.
- additives include buffers, carriers, stabilizers, excipients, preservatives, tonicity adjusting agents, anti-oxidants and the like (e.g ., viscosity adjusting agents or extenders).
- the selection of specific additives will depend upon the storage form (i.e., liquid or lyophilized) and the modes of administering the KGF analog. Suitable formulations, known in the art, can be found in REMINGTON'S PHARMACEUTICAL SCIENCES (latest edition), Mack Publishing Company, Easton, PA.
- the KGF analogs may be applied in therapeutically effective amounts to tissues specifically characterized by having damage to or clinically insufficient numbers of non-fibroblast epithelium cells. Since KGF binds to heparin, it is likely that heparin, heparin sulfate, heparin-like glycosaminglycans and heparin-like glycosaminoglycans, which are present in the extracellular environment may bind KGF in vivo . It follows that KGF analogs with reduced heparin binding ability will have enhanced potency, as more KGF will reach its targeted receptor and will not be sequestered by heparin and heparin-like compounds in the extracellular environment. These analogs will be more useful therapeutically, as lower dosages of a particular KGF analog will be required per treatment.
- the KGF analogs may be applied in therapeutically effective amounts to tissues specifically characterized by having damage to or clinically insufficient numbers of non-fibroblast epithelium cells. Areas in which KGF analogs may be successfully administered include, but are not limited to: the stimulation, proliferation and differentiation of adnexal structures such as hair follicles, sweat glands, and sebaceous glands in patients with burns and other partial and full-thickness injuries; accelerated reepithelialization of lesions caused by epidermolysis bullosa, which is a defect in adherence of the epidermis to the underlying dermis, resulting in frequent open, painful blisters which can cause severe morbidity; preventing chemotherapy-induced alopecia and treating male-pattern baldness, or the progressive loss of hair in men and women; treating gastric and duodenal ulcers; treating inflammatory bowel diseases, such a Crohn's disease (affecting primarily the small intestine) and ulcerative colitis (affecting primarily the large bowel); preventing
- a patient in need of proliferation of non-fibroblast epithelial cells will be administered an effective amount of a KGF analog.
- An "effective amount” is that amount of KGF analog required to elicit the desired response in the patient being treated and will, thus, generally be determined by the attending physician.
- Factors influencing the amount of KGF analog administered will include the age and general condition of the patient, the disease being treated, etc. Typical dosages will range from 0.001 mg/kg body weight to 500 mg/kg body weight.
- the KGF analog may be safely administered parenterally (e.g., via IV, IT, IM, SC, or IP routes), orally or topically to warm-blooded animals (such as humans).
- the KGF analog may be used once or administered repeatedly, depending on the disease and condition of the patient. In some cases, the KGF analog may be administered as an adjunct to other therapy and also with other pharmaceutical preparations.
- RNA isolated from cells known to produce the polypeptide was performed. Initially, cells from a human fibroblast cell line AG1523A (obtained from Human Genetic Mutant Cell Culture Repository Institute For Medical Research, Camden, New Jersey) were disrupted with guanidium thiocyanate, followed by extraction (according to the method of Chomyzinski et al. (1987), Anal. Biochem., 172 :156). Using a standard reverse transcriptase protocol for total RNA, the KGF cDNA was generated.
- AG1523A obtained from Human Genetic Mutant Cell Culture Repository Institute For Medical Research, Camden, New Jersey
- PCR (PCR#1) amplification of the KGF gene was carried out using the KGF cDNA as template and primers OLIGO#1 and OLIGO#2 that encode DNA sequences immediately 5' and 3' of the KGF gene [Model 9600 thermocycler (Perkin-Elmer Cetus, Norwalk, CT); 28 cycles; each cycle consisting of one minute at 94°C for denaturation, two minutes at 60°C for annealing, and three minutes at 72°C for elongation].
- a small aliquot of the PCR#1 product was then used as template for a second KGF PCR (PCR#2) amplification identical to the cycle conditions described above except for a 50°C annealing temperature.
- PCR#3 and OLIGO#4 were used to modify the KGF DNA product from PCR#2 to include MluI and BamHI restriction sites at the 5' and 3' ends of the gene, respectively [PCR#3; 30 cycles; each cycle consisting of one minute at 94°C for denaturation, two minutes at 60°C for annealing, and three minutes at 72°C for elongation].
- This DNA was subsequently cut with MluI and BamHI , phenol extracted, and ethanol precipitated. It was then resuspended and ligated (using T4 ligase) into a pCFM1156 plasmid ( Figure 2A) that contained a "RSH" signal sequence to make construct RSH-KGF ( Figure 3).
- the ligation products were transformed (according to the method of Hanahan (1983), J. Mol. Biol., 166 :557) into E. coli strain FM5 (ATCC: 53911) and plated onto LB+kanamycin at 28°C. Several transformants were selected and grown in small liquid cultures containing 20 ⁇ g/mL kanamycin.
- the RSH-KGF plasmid was isolated from the cells of each culture and DNA sequenced. Because of an internal NdeI site in the KGF gene, it was not possible to directly clone the native gene sequence into the desired expression vector with the bracketed restriction sites of NdeI and BamHI. This was accomplished as a three-way ligation.
- Plasmid RSH-KGF was cut with the unique restriction sites of BsmI and SstI , and a ⁇ 3 kbp DNA fragment (containing the 3' end of the KGF gene) was isolated following electrophoresis through a 1% agarose gel.
- a PCR (PCR#4) was carried out as described for PCR#3 except for the substitution of OLIGO#5 for OLIGO#3.
- the PCR DNA product was then cut with NdeI and BsmI and a 311 bp DNA fragment was isolated following electrophoresis through a 4% agarose gel.
- the third piece of the ligation is a 1.8 kbp DNA fragment of pCFM1156 cut with NdeI and SstI which was isolated following electrophoresis through a 1% agarose gel.
- T4 ligase T4 ligase
- transformation, kanamycin selection and DNA sequencing as described above a clone was picked containing the construct in Figure 4 and the plasmid designated KGF.
- KGF DNA sequence between the unique KpnI and EcoRI sites was replaced with chemically synthesized OLIGOs (OLIGO#6 through OLIGO#11) to minimize the use of the internal start site ( Figure 5).
- the OLIGOs were phosphorylated with T4 polynucleotide kinase and then heat denatured.
- the single-stranded (ss) OLIGOs were then allowed to form a ds DNA fragment by allowing the temperature to slowly decrease to room temperature.
- T4 ligase was then used to covalently link both the internal OLIGO sticky-ends and the whole ds OLIGO fragment to the KGF plasmid cut with KpnI and EcoRI.
- the new plasmid was designated KGF(dsd).
- a completely E. coli codon-optimized KGF gene was constructed by PCR amplification of chemically synthesized OLIGOs #12 through 24.
- OLIGOs #12 through 24 were designed so that the entire DNA sequence encoding native KGF was represented by OLIGOs from either the "Watson” or the "Crick” strand and upon PCR amplification would produce the desired double-stranded DNA sequence ( Figure 6) [PCR#5, Model 9600 thermocycler, Perkin-Elmer Cetus]; 21 cycles, each cycle consisting of 31 seconds at 94°C for denaturation, 31 seconds at 50°C for annealing, and 31 seconds at 73°C for elongation; following the 21 cycles the PCR was finished with a final elongation step of 7 minutes].
- PCR#5 utilized the outside primers (100 pmoles/100 ⁇ l rxn) OLIGO#12 and OLIGO#13 and 1 ⁇ l/100 ⁇ l rxn of a KGF template derived by ligation (by T4 ligase) of OLIGO #14 through OLIGO#19 (OLIGO#15 through OLIGO#18 were phosphorylated with T4 polynucleotide kinase) using OLIGO#20 through OLIGO#24 as band-aid oligos (Jayaraman et al. (1992), Biotechniques, 12 :392) for the ligation.
- KGF codon optimized
- KGF analogs described herein are composed in part from DNA sequences found in KGF(dsd) or KGF(codon optimized), or a combination of the two.
- the sequences are further modified by the insertion into convenient restriction sites of DNA sequences that encode the particular KGF analog amino acids made utilizing one or more of the above-described techniques for DNA fragment synthesis. Any of the analogs can be generated in their entirety by the above described techniques.
- E. coli codons were used where appropriate, although the presence of E. coli optimized codons in part or in toto of any of the genes where examined did not significantly increase the yield of protein that could be obtained from cultured bacterial cells.
- FIGS 7 to 10 and 17 to 26 set forth by convenient example particular KGF analog nucleotide and amino acid sequence constructions: R(144)Q ( Figure 7); C(1,15)S/R(144)E ( Figure 8); C(1,15)S/R(144)Q (Figure 9); ⁇ N23/R(144)Q ( Figure 10); ⁇ N23/N(137)E ( Figure 17); ⁇ N23/K(139)E ( Figure 18); ⁇ N23/K(139)Q ( Figure 19); ⁇ N23/R(144)A ( Figure 20); ⁇ N23/R(144)L ( Figure 21); ⁇ N23/K(147)E ( Figure 22); ⁇ N23/K(147)Q (Figure 23); ⁇ N23/K(153)E ( Figure 24); ⁇ N23/K(153)Q; ( Figure 25) and ⁇ N23/Q(152)E/K(153)E ( Figure 26). All the KGF analog constructions described herein were DNA sequence confirmed.
- the plasmid p3102 can be derived from the plasmid pCFM1656 by making a series of site-directed base changes with PCR overlapping oligo mutagenesis. Starting with the BglII site (pCFM1656 plasmid bp # 180) immediately 5' to the plasmid replication promoter, PcopB, and proceeding toward the plasmid replication genes, the base pair changes are as follows:
- pCFM1156, pCFM1656 and pCFM3102 are very similar to each other and contain many of the same restriction sites.
- the plasmids were chosen by convenience, and the vector DNA components can be easily exchanged for purposes of new constructs.
- the host used for all cloning was E. coli strain FM5 (ATCC: 53911) and the transformations were carried out (according to the method of Hanahan (1983), supra) or by electroelution with a Gene PulserTM transfection apparatus (BioRad Laboratories, Inc., Hercules, CA) according to the manufacturer's instructions.
- Feed batch fermentation starts with the feeding of Feed # 1 medium (Tsai, et al. (1987), supra).
- Feed # 1 medium (Tsai, et al. (1987), supra).
- OD600 OD600
- Feed 1 was discontinued in favor of Feed 2, the addition rate of which was initiated at 300 mL/hr.
- Feed 2 comprised 175 g/L trypticase-peptone, 87.5 g/L yeast extract, and 260 g/L glucose.
- the culture temperature was decreased to 36°C, where this temperature was then maintained for another 6 hours.
- the fermentation was then halted and the cells were harvested by centrifugation into plastic bags placed within 1 L centrifuge bottles.
- the cells were pelleted by centrifugation at 400 rpm for 60 minutes, after which the supernatants were removed and the cell paste frozen at -90°C.
- native KGF, R(144)Q, C(1,15)S/R(144)E, C(1,15)S/R(144)Q and ⁇ N23/R(144)Q proteins were purified using the following procedure.
- Cell paste from a high cell density fermentation was suspended at 4°C in 0.2 M NaCl, 20 mM NaPO 4 , pH 7.5 as a 10-20% solution (weight per volume) using a suitable high shear mixer.
- the suspended cells were then lysed by passing the solution through a homogenizer (APV Gaulin, Inc., Everett, MA) three times.
- the outflowing homogenate was cooled to 4-8°C by using a suitable heat exchanger.
- Debris was then removed by centrifuging the lysate in a J-6BTM centrifuge (Beckman Instruments, Inc., Brea, CA) equipped with a JS 4.2 rotor at 4,200 rpm for 30-60 min. at 4°C. Supernatants were then carefully decanted and loaded onto a previously prepared 450 mL (5 cm x 23 cm) column of S-Sepharose Fast FlowTM resin (Pharmacia, Piscataway, NJ) equilibrated with 0.2 M NaCl, 20 mM NaPO 4 , pH 7.5 at 4°C.
- the column was washed with five column volumes (2250 mL) of 0.4 M NaCl, 20 mM NaPO 4 , pH 7.5 at 4°C.
- the desired protein was eluted by washing the column with 5 L of 0.5 M NaCl, 20 mM NaPO 4 , pH 7.5.
- 50 mL fractions were collected and the A 280 of the effluent was continuously monitored. Fractions identified by A 280 as containing eluted material were then analyzed by SDS-PAGE through 14% gels to confirm the presence of the desired polypeptide.
- fractions were collected under constant A 280 monitoring of the effluent. Those fractions containing the protein (determined by 14% SDS-PAGE) were then pooled, followed by concentration through a YM-10 membrane (10,000 molecular weight cutoff) in a 350cc stirring cell (Amicon, Inc. Mayberry, MA) to a volume of 30-40 mL.
- the concentrate was then loaded onto a previously generated 1,300 mL (4.4 cm x 85 cm) column of Superdex-75TM resin (Pharmacia) equilibrated in column buffer comprising 1X PBS (Dulbecco's Phosphate Buffered Saline, "D-PBS", calcium and magnesium-free) or 0.15 M NaCl, 20 mM NaPO 4 , pH 7.0.
- 1X PBS Dulbecco's Phosphate Buffered Saline, "D-PBS", calcium and magnesium-free
- 0.15 M NaCl 20 mM NaPO 4 , pH 7.0.
- the protein was eluted from the gel filtration matrix using column buffer. Thereafter, 10 mL fractions were recovered and those containing the analog (determined by 14% SDS-PAGE) were pooled.
- the protein concentration was about 5-10 mg/mL in the resultant pool. All of the above procedures were performed at 4-8°C, unless otherwise specified.
- the polypeptides were compared by their storage stability, thermal unfolding transition temperatures (T m ), and stability in a broad range of pH conditions.
- Visible precipitates were removed by centrifuging 250 ⁇ L of each sample through a 0.22 ⁇ m Spin-X filter unit (Costar, Cambridge, MA). Soluble protein in the filtered solutions was subsequently analyzed by size exclusion HPLC. The amount of soluble protein was determined by integrating the HPLC peak area and plotting the result as a function of incubation time at 37°C.
- the results of native KGF, C(1,15)S, C(1,15)S/R(144)Q, and C(1, 15)S/R(144)E are shown in Figure 11. The data for R(144)Q and ⁇ N23/E(144)Q are not shown.
- Thermal unfolding was monitored by circular dichroism (CD) at 230 nm using a J-720TM spectropolarimeter (Jasco, Inc., Easton, MD) equipped with a PTC-343 Peltier-type temperature control system.
- CD analysis separate samples containing 0.1 mg/mL of the polypeptide to be analyzed were prepared in D-PBS (Life Technologies, Inc., Grand Island, NY). For each sample, about 2.5 mL was loaded into a 10 mm path length rectangular SuprasilTM quartz (Heraeus Quarzschmelze, GmbH, Hanau, Germany) fluorescent cell (Hellma Cells, Inc., Jamaica, NY). The cell was then placed into the Peltier-type temperature control system in the spectropolarimeter.
- Thermal unfolding was carried out at a rate of 50°C/hr. Changes in ellipticity were monitored at 230 nm to indicate unfolding.
- the T m of each sample was estimated by identifying a temperature at which 50% of protein molecules in the solution were unfolded ( Biophysical Chemistry, Cantor and Schimmel (eds), W.H. Freeman and Co. San Francisco (1980)). The estimated T m for each of the five proteins is listed in Table 2.
- the acid stabilities of C(1,15)S/R(144)Q, C(1,15)S, and C(1,15)S/R(144)E were also compared to that of native KGF, by adjusting D-PBS to different pH values by adding concentrated HCl or NaOH. Approximately 2.35 mL of D-PBS at different pH values was mixed with 100 ⁇ L of 2.45 mg/mL KGF protein in a quartz cell. These samples were thermally unfolded at a rate of 50°C/hr and monitored by CD at 230 nm.
- Figure 12 shows the T m as a function of pH for native KGF, C(1,15)S/R(144)Q and C(1,15)S/R(144)E. In the pH range tested, the C(1,15)S/R(144)Q and C(1,15)S/R(144)E always have a higher T m than the native KGF.
- the concentrations of each of the KGF analogs relative to a known standard native KGF was determined using an in vitro biological assay. Each KGF analog was then diluted and assayed for biological activity using a Balb/MK mitogenic assay. The samples were first diluted in a bioassay medium consisting of 50% customer-made Eagle's MEM, 50% customer-made F12, 5 ⁇ G/mL transferrin, 5 ng/ml sodium selenite, 0.0005% HSA and 0.005% Tween 20. KGF samples were then added into Falcon Primeria 96-well plates seeded with Balb/MK cells.
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Claims (19)
- Ein Polypeptidanalogon des als "KGF' bezeichneten nativen Keratinozytenwachstumsfaktors, wobei der native KGF mit der folgenden Sequenz korrespondiert: wobei besagtes Analogon eine erhöhte Stabilität aufweist, wobei das Analogon die Aminosäuresequenz von KGF umfasst, die einen Ladungswechsel durch die Deletion und/oder Substitution von einem oder mehreren der Aminosäurereste 41 - 154 der oben angegebenen Sequenz aufweist, wodurch eine verringerte positive Ladung im Vergleich zu nativem KGF bewirkt wird, wobei dieses optional zusätzlich eine Deletion der ersten 15 bis 24 Aminosäuren des N-terminalen nativen KGF, der zu Cys (1) und Cys (15) korrespondierende ersetzte oder deletierte Reste aufweist, ein N-terminales Methionin aufweist oder eine Signalsequenz aufweist, mit der Proviso, dass die Aminosäuresequenz 123-131 der oben angegebenen Sequenz nicht durch eine der Aminosäuresequenzen, ausgewählt aus der Gruppe DLYQG und AKYEG, substituiert ist.
- Das Polypeptidanalogon gemäß Anspruch 1, wobei die deletierten oder substituierten Aminosäurereste ausgewählt sind aus dem Argininenrest in der Aminosäureposition 41, dem Glutaminrest in der Aminosäureposition 43, dem Lysinrest in der Aminosäureposition 55, dem Lysinrest in der Aminosäureposition 95, dem Asparaginrest in der Aminosäureposition 137, dem Glutaminrest in der Aminosäureposition 138, dem Lysinrest in der Aminosäureposition 139, dem Argininrest in der Aminosäureposition 144, dem Lysinrest in der Aminosäureposition 147, dem Glutaminrest in der Aminosäureposition 152, dem Lysinrest in der Aminosäureposition 153 oder dem Threoninrest in der Aminosäureposition 154 der in Anspruch 1 gezeigten Sequenz.
- Das Polypeptidanalogon gemäß Anspruch 1, ausgewählt aus R(144)Q; C(1,15)S/R(144)E; C(1,5)S/R(144)Q; ΔN23/R(144)Q; ΔN23/N(137)E; ΔN23/K(139)E; ΔN23/K(139)Q; ΔN23/R(144)A; ΔN23/R(144)E; ΔN23/R(144)L; ΔN23/K(147)E; ΔN23/K(147)Q; ΔN23/K(153)E; ΔN23/K(153)Q; oder ΔN23/Q(152)E/K(153)E.
- Das Polypeptidanalogon gemäß einem der Ansprüche 1 bis 3, wobei besagtes Polypeptidanalogon kovalent an einen chemischen Bereich gebunden ist.
- Das Polypeptidanalogon gemäß Anspruch 4, wobei besagter chemischer Bereich Polyethylenglycol ist.
- Das Polypeptidanalogon gemäß einem der Ansprüche 1 bis 5, wobei besagtes Polypeptidanalogon lyophilisiert ist.
- Eine pharmazeutische Formulierung, umfassend eine therapeutisch wirksame Menge eines Polypeptidanalogon gemäß einem der Ansprüche 1 bis 6 und einen pharmazeutisch verträglicher Träger.
- Ein rekombinantes Nukleinsäuremolekül, dass ein Polypeptidanalogon gemäß einem der Ansprüche 1 bis 3 kodiert.
- Ein biologisch funktionelles Plasmid oder viraler Vector, umfassend ein rekombinantes Nukleinsäuremolekül gemäß Anspruch 8.
- Eine prokariontische oder eukariontische Wirtszelle, enthaltend eine rekombinante Nukleinsäure gemäß Anspruch 8 oder einen biologisch funktionalen Vector gemäß Anspruch 9.
- Eine prokariontische Wirtszelle gemäß Anspruch 10, die E. coli ist.
- Eine eukariontische Wirtszelle gemäß Anspruch 10, die eine Säugetierzelle ist, vorzugsweise eine chinesische Hamsterovarzelle.
- Ein Verfahren für die Herstellung eines Polypeptidanalogons gemäß einem der Ansprüche 1 bis 6, wobei das Verfahren das Wachsen einer prokariontischen oder eukariontischen Wirtszelle gemäß einem der Ansprüche 10 bis 12 unter geeigneten Nährstoffbedingungen in einer Weise umfasst, die die Expression des kodierten Polypeptidanalogons zulässt und die Isolierung des so hergestellten Polypeptidanalogons.
- Ein in vitro-Verfahren zur Stimulation der Produktion von Nichtfibroblasten-Epithelzellen, umfassend das in Kontaktbringen solcher Zellen mit einer wirksamen Menge eines Peptidanalogons gemäß einem der Ansprüche 1 bis 6 oder einer pharmazeutischen Formulierung gemäß Anspruch 7.
- Verwendung eines Polypeptidanalogons gemäß einem der Ansprüche 1 bis 6 für die Herstellung einer pharmazeutischen Formulierung für die Behandlung von Krankheiten, die die Stimulation der Produktion von Nichtfibroblasten-Epithelzellen benötigen.
- Eine Verwendung einer wirksamen Menge eines Polypeptidanalogons gemäß einem der Ansprüche 1 bis 6 für die Herstellung eines Medikaments zur Stimulation von Nichtfibroblasten-Epithelzellen in einem Patienten, der dessen bedarf.
- Die Verwendung gemäß Anspruch 16, wobei besagte Nichtfibroblasten-Epithelzellen ausgewählt sind aus adnexalen Strukturen, Leberzellen, Mukosaepithel in den respiratorischen und gastrointestinalen Trakten, komealen Zellen oder tympanische Epithelzellen.
- Eine Verwendung einer wirksamen Menge des Polypeptidsanalogons gemäß einem der Ansprüche 1 bis 6 für die Herstellung eines Medikaments zur Stimulation der Produktion vom Nichtfibroblasten-Epithelzellen in einem Patienten für die Prävention oder Behandlung eines Zustandes, wobei besagter Zustand ausgewählt ist aus Verbrennungen oder anderen Verletzungen teilweiser oder vollständiger Dicke; Epidermolyse bullosa; Chemotherapie-indizierter Alopecia; männliche Musterglatzköpfigkeit; progressiver Verlust von Haar in Männern und Frauen; Magen- und Zwölffingerdarmgeschwüre; entzündliche Darmkrankheiten wie Crohn's-Krankheit und geschwürartiger Darmkatarrh; Darmtoxizität bei Strahlungs- und Chemotherapiebehandlungen; Hyaline Membrankrankheit; akuter oder chronischer Lungenschaden; Leberzirrhose, fulminantes Leberversagen, akute virale Hepatitis und/oder toxische Angriffe auf die Leber; komeale Abschürfungen; progressive Zahnfleischerkrankung oder Trommelfellschaden.
- Ein Kit, umfassend ein Polypeptidanalogon gemäß einem der Ansprüche 1 bis 6 oder eine pharmazeutische Formulierung von Anspruch 7.
Priority Applications (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
SI9530653T SI0785950T1 (en) | 1994-10-13 | 1995-10-12 | Keratinocyte growth factor analogs |
Applications Claiming Priority (5)
Application Number | Priority Date | Filing Date | Title |
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US32333794A | 1994-10-13 | 1994-10-13 | |
US323337 | 1994-10-13 | ||
US48782595A | 1995-06-07 | 1995-06-07 | |
US487825 | 1995-06-07 | ||
PCT/US1995/013075 WO1996011951A2 (en) | 1994-10-13 | 1995-10-12 | Keratinocyte growth factor analogs |
Publications (2)
Publication Number | Publication Date |
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EP0785950A2 EP0785950A2 (de) | 1997-07-30 |
EP0785950B1 true EP0785950B1 (de) | 2003-04-16 |
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Application Number | Title | Priority Date | Filing Date |
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EP95938216A Expired - Lifetime EP0785950B1 (de) | 1994-10-13 | 1995-10-12 | Analogen des keratinozytenwachstumfaktors |
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Country | Link |
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EP (1) | EP0785950B1 (de) |
JP (2) | JP4422210B2 (de) |
AT (1) | ATE237634T1 (de) |
AU (1) | AU3893195A (de) |
CA (1) | CA2202390C (de) |
DE (1) | DE69530404T2 (de) |
DK (1) | DK0785950T3 (de) |
ES (1) | ES2196088T3 (de) |
HU (1) | HUT78069A (de) |
MX (1) | MX9702665A (de) |
NO (1) | NO971621L (de) |
PT (1) | PT785950E (de) |
SI (1) | SI0785950T1 (de) |
WO (1) | WO1996011951A2 (de) |
Families Citing this family (16)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
ES2227527T3 (es) | 1993-06-29 | 2005-04-01 | Chiron Corp | Un factor de crecimiento de queratinocitos (kgf) truncado que tiene actividad biologica aumentada. |
US7084119B2 (en) | 1993-06-29 | 2006-08-01 | Chiron Corporation | Truncated keratinocyte growth factor (KGF) having increased biological activity |
CZ98297A3 (cs) * | 1994-10-13 | 1998-08-12 | Amgen Inc. | Způsob purifikace keratinocytových růstových faktorů |
US6077692A (en) | 1995-02-14 | 2000-06-20 | Human Genome Sciences, Inc. | Keratinocyte growth factor-2 |
US7232667B2 (en) | 1995-02-14 | 2007-06-19 | Human Genome Sciences, Inc. | Keratinocyte growth factor-2 polynucleotides |
US6693077B1 (en) | 1995-02-14 | 2004-02-17 | Human Genome Sciences, Inc. | Keratinocyte growth factor-2 |
AU720232B2 (en) | 1996-07-19 | 2000-05-25 | Amgen, Inc. | Analogs of cationic proteins |
EP1473366A1 (de) * | 1996-10-15 | 2004-11-03 | Amgen Inc. | Produkte des Keratinocyten-Wachstumsfaktors 2 |
DK0935652T3 (da) * | 1996-10-15 | 2004-07-26 | Amgen Inc | Keratinocyt vækstfaktor-2 produkter |
US6743422B1 (en) | 1996-10-15 | 2004-06-01 | Amgen, Inc. | Keratinocyte growth factor-2 products |
EP1041996A4 (de) * | 1997-12-22 | 2003-05-14 | Human Genome Sciences Inc | Keratinozyten wachstumsfaktor-2 formulierungen |
US6869927B1 (en) | 1997-12-22 | 2005-03-22 | Human Genome Sciences, Inc. | Keratinocyte growth factor-2 formulations |
US6248725B1 (en) | 1999-02-23 | 2001-06-19 | Amgen, Inc. | Combinations and methods for promoting in vivo liver cell proliferation and enhancing in vivo liver-directed gene transduction |
MXPA04001526A (es) | 2001-08-21 | 2004-05-31 | Chiron Corp | Composiciones de polipetidos kgf. |
BRPI0519070A2 (pt) | 2004-12-15 | 2008-12-23 | Amgen Inc | composiÇço de fator de crescimento de queratinàcito liofilizada, mÉtodos para preparar um fator de crescimento de queratinàcito liofilizado, e para tratar uma doenÇa, e, kit para preparar uma composiÇço farmacÊutica aquosa |
KR20230127721A (ko) | 2022-02-25 | 2023-09-01 | 한국해양과학기술원 | 온도안정성을 향상시킨 fgf7 폴리펩타이드 및 그 용도 |
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ATE197800T1 (de) * | 1989-01-31 | 2000-12-15 | Jeffrey S Rubin | Dns kodierend für einen für epithelzellen spezifischen wachstumsfaktor |
CA2172137C (en) * | 1993-09-24 | 1999-12-14 | Andrew Peter Seddon | Surface loop structural analogues of fibroblast growth factors |
-
1995
- 1995-10-12 WO PCT/US1995/013075 patent/WO1996011951A2/en active IP Right Grant
- 1995-10-12 AU AU38931/95A patent/AU3893195A/en not_active Abandoned
- 1995-10-12 EP EP95938216A patent/EP0785950B1/de not_active Expired - Lifetime
- 1995-10-12 AT AT95938216T patent/ATE237634T1/de active
- 1995-10-12 CA CA002202390A patent/CA2202390C/en not_active Expired - Fee Related
- 1995-10-12 DE DE69530404T patent/DE69530404T2/de not_active Expired - Lifetime
- 1995-10-12 HU HU9901325A patent/HUT78069A/hu unknown
- 1995-10-12 DK DK95938216T patent/DK0785950T3/da active
- 1995-10-12 JP JP51337096A patent/JP4422210B2/ja not_active Expired - Fee Related
- 1995-10-12 SI SI9530653T patent/SI0785950T1/xx unknown
- 1995-10-12 MX MX9702665A patent/MX9702665A/es not_active IP Right Cessation
- 1995-10-12 PT PT95938216T patent/PT785950E/pt unknown
- 1995-10-12 ES ES95938216T patent/ES2196088T3/es not_active Expired - Lifetime
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1997
- 1997-04-09 NO NO971621A patent/NO971621L/no unknown
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Also Published As
Publication number | Publication date |
---|---|
ES2196088T3 (es) | 2003-12-16 |
WO1996011951A2 (en) | 1996-04-25 |
NO971621L (no) | 1997-06-12 |
PT785950E (pt) | 2003-09-30 |
EP0785950A2 (de) | 1997-07-30 |
JP2007020575A (ja) | 2007-02-01 |
DK0785950T3 (da) | 2003-07-28 |
CA2202390A1 (en) | 1996-04-25 |
SI0785950T1 (en) | 2003-08-31 |
JP2000511762A (ja) | 2000-09-12 |
CA2202390C (en) | 2005-05-17 |
MX9702665A (es) | 1997-07-31 |
DE69530404D1 (de) | 2003-05-22 |
DE69530404T2 (de) | 2003-11-13 |
HUT78069A (hu) | 1999-08-30 |
JP4422210B2 (ja) | 2010-02-24 |
ATE237634T1 (de) | 2003-05-15 |
NO971621D0 (no) | 1997-04-09 |
AU3893195A (en) | 1996-05-06 |
WO1996011951A3 (en) | 1996-12-12 |
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